Most biological cells can drastically change their shape (called cell deformability) without a change in the functional integrity of the cell membrane. Under certain conditions, the cell membrane can rupture to lose cell contents (called cell fragility). Cells can have any combination of a high-to-low deformability and a high-to-low fragility. However, most cells normally have high deformability and moderate-to-low fragility.
Deformability is an important characteristic for function of some normally stationary cells such as "sensory receptors" and normally moving cells such as blood and lymph cells. Cell fragility is an important characteristic for almost all body cells because changes in the cell environment can shift water into a cell to rupture fragile cells. Thus, changes in cell fragility can also change the ability of a cell to perform its normal function. For example, an abnormally high fragility to rupture part of the circulating pool of red blood cells will reduce the transport of oxygen to tissues. Thus, fragility might clinically be a more important cell characteristic than deformability.
Cell fragility changes with cell age, duration of blood-bank storage, treatment with a variety of membrane-binding drugs, and progression of membrane or hemoglobin-related diseases such as sickle-cell anemia and diabetes. Thus, a rapid, highly accurate, and easily applied method is needed for clinical measurements to assess cell fragility as an index of cell ability to function (called cell integrity). The current methods use mechanical forces primarily to assess the deformability or fragility of red blood cells. These current methods include Osmotic-Gradient Ektacytometry, Filtration, Micropipette Suction, and Osmotic Fragility. Of these, only the Osmotic Fragility method is commonly used in clinical laboratories.
Osmotic-Gradient Ektacytometry is one technology that has been developed to measure cell deformability. This technology uses a viscometer to measure shape changes which are induced in red blood cells by different osmotic solutions at various levels of rotational speed (called applied shear stress). The use of different osmotic solutions offers the potential for this Ektacytometry method to determine several properties which could influence the deformability of red blood cells. However, the osmotic-spectrum curves which are produced by Ektacytometry are complex and very difficult to interpret. These curves are also subject to high variability due to changes in sample ambient temperature, pH, and plasma osmolality. Thus, Ektacytometry currently requires very sophisticated equipment, extensive operator training, and highly controlled test conditions to make it usable, but only in a few research laboratories and not in the clinical setting.
Other methods for measuring cell deformability include filtration through various size pores, aspiration of cells into micropipettes of fixed tip size and taper, and a photometric technique. All of these methods also require very sophisticated equipment and substantial operator training; and they are extremely time consuming for analysis of relatively few cells in a few samples. Thus, these methods have also not been accepted into general clinical use.
Impedance measurements (Hands et al, U.S. Pat. No. 4,835,457) and time measurements (David D. Paterson, U.S. Pat. No. 4,491,012) have been made on red cells that pass under pressure through a membrane or a foil system (Helmut Jahn, U.S. Pat. No. 4,797,606) as variations of the Filtration method for measurement of cell deformability. These variations are extremely sensitive to manufacturing tolerances on the filter or foil and they primarily measure only deformability rather than cell fragility. Thus, these variant Filtration methods have also not been accepted into common clinical use.
The Osmotic Fragility test was one of the earliest methods that was developed for assessment of red blood cell integrity, and it is one of the few tests currently in clinical use. The Osmotic Fragility test is time-consuming, requires multiple blood handling steps, usually requires relatively large blood samples, and provides no information about the lysis (cell membrane disruption) rate. These limitations and the lack of sensitivity to mild or moderate changes in cell fragility has often led to the clinical use of this osmotic test only for diagnosis of one disease called hereditary spherocytosis.
It was first shown some fifty years ago that some chemicals can be changed by light (called photoactivation) to induce the rupture or breakup of red blood cells (called hemolysis) in a test tube. Since then, this basic process (called photohemolysis) has been extensively studied in a variety of test-tube experiments.
The mechanism for photohemolysis is oxygen dependent and probably involves the generation of singlet oxygen with the subsequent oxidation of proteins in the red blood cell membrane. This protein oxidation leads to the creation of water channels with an increase in passive cationic exchange and the subsequent influx of water into the cell to produce hemolysis. Photohemolysis could also involve peroxidation of the lipid layers of the cell membrane. This peroxidation would alter membrane fluidity in the lipid bilayer to limit the ability of the cell to undergo shape changes, which are dependent on changes in the lipid bilayer.
Thus, there is considerable scientific evidence to show that certain chemical agents can be photoactivated to disrupt red blood cells by altering either the protein or the lipid layer of the cell membrane. These membrane alterations disrupt membrane integrity to permit water inflow which changes cell shape (called cell deformability) by increasing cell volume until the "internal cell pressure" breaks the cell membrane (called cell fragility) sufficiently to permit loss of cell contents (called lysis or hemolysis in the case of red blood cells).
Current clinical methods use milliliter quantities of blood solutions to measure the osmolality (equivalent to internal water pressure) at which hemolysis occurs. However, these hemolysis methods cannot separate changes in deformability from those in fragility, cannot distinguish between loss of membrane integrity due to protein layer changes and that due to lipid layer changes, and cannot determine "rates of hemolysis" to provide a more sensitive hemolysis test for clinical use.
Current clinical methods are "macro" techniques in that relatively large blood volumes (milliliter quantities) are exposed to various osmotic solutions to determine hemolysis. These Osmotic Fragility tests are generally performed in relatively large test tubes or cuvettes using parallel light for activation. Likewise, current photohemolysis research methods are also "macro" techniques in that milliliter quantities of blood are exposed to photoactivation, and long analysis times are required to obtain a single measurement. Photoactivation of red blood cells incubated with 0.1 mM of protoporphyrin as a "cell-attack" agent requires about twenty minutes of illumination time to achieve a modest 20% hemolysis while a 100% hemolysis requires a 25-minute or longer exposure. Similarly, more than twenty minutes of light exposure is needed with pheophorbide as the cell-attack agent to give about a 90% hemolysis. Light exposures of 3-4 hours are needed with eosin-isothiocyanate as the cell-attack agent to give maximal hemolysis which occurs about 11 hours after photoactivation. All of these current methods require extended time periods primarily because they use unfocused light which limits the activation to low light power densities.
All of the current Osmotic Fragility and Photohemolysis methods require multiple dilutions, centrifugation, and analysis in a spectrophotometer to give single measurements of a percent cell hemolysis. Thus, the current clinical and research methods are time consuming, and simultaneous light-dose and time-dependent relationships are next-to-impossible to obtain from one blood sample. Some researchers have used a light scattering device to detect photohemolysis. However, this device is very sensitive to very small changes in red blood cell concentrations, and this device requires a monolayer of red blood cells with a red cell concentration of less than 0.00025% which is extremely difficult to achieve even by current micropipetting systems. Even then, this cell monolayer method is quite time-consuming since 100% hemolysis requires 4 hours with phloxinc B as the cell-attack agent.